Project Summary A diversity of tolerance mechanisms work together to prevent autoimmune disease in most individuals by limiting the activity of self-reactive lymphocytes. How genetically determined defects in particular tolerance pathways may interact to lead to autoimmune disease is not well understood. The goal of this project is to provide insights into the basis of multigenic autoimmune susceptibility by modeling it in mice using two genetic defects with especially well understood immunologic mechanisms, namely a complete deficiency in Lyn and partial deficiency in Aire. Aire controls the expression of a large number of tissue-restricted self antigens in the thymus and therefore promotes central tolerance of T cells. Lyn is a protein tyrosine kinase that restrains the activities of dendritic cells, macrophages, and B cells due to its importance in mediating the signaling of inhibitory receptors on the surface of these cells. Remarkably, mice containing both a hypomorphic allele of Aire, which by itself leads to little autoimmunity on the autoimmune resistant C57BL/6 background, and a deficiency in Lyn spontaneously develop a highly destructive autoimmune attack on their retinas. This disease occurs in 50% of Lyn-/- AireGW/+ mice, all of which have severe disease by 8 weeks of age, whereas the other 50% are protected. The disease prevalence in the double mutant mice is strikingly similar to results of identical twin studies in humans with autoimmune diseases. Moreover, this new animal model of spontaneous organ- specific autoimmune disease has several features that are especially favorable for mechanistic dissection and may make it possible to provide new conceptual insights into the nature of autoimmune disease susceptibility and initiation. The proposed studies will determine the epitope specificity and avidity of the CD4 T cells that break tolerance and initiate autoimmune attack. How reduced self-antigen expression in the thymus enables the pathogenic potential of these T cells will be determined. In addition, for mice that do not develop uveitis, there is a small expansion of retinal autoantigen-specific CD4 T cells in the eye-draining lymph nodes. The immunological mechanisms that prevent these T cells from initiating autoimmune attack in 50% of animals will be determined. We shall determine whether tolerance in the protected animals is primarily due to anergy of effector T cells, to dominant suppression by retina-specific Treg, or to a tissue-specific checkpoint. In these protected mice, we shall determine whether immunological perturbations can unleash disease in otherwise protected animals, a situation that may be relevant to the organ-specific autoimmunity seen in some cancer patients treated with checkpoint blockade. These studies will provide novel and valuable insights into the question of how genetic defects in two different immune tolerance pathways can interact to result in genetic susceptibility to autoimmune disease and in understanding how autoimmune disease in initiated in some but not all individuals with the same degree of genetic susceptibility.